ABSTRACT The human malaria parasite Plasmodium falciparum imports and catabolizes massive amounts of host erythrocyte cytosol (mainly hemoglobin) during its asexual replication cycle. The mechanism and regulation of hemoglobin uptake, which occurs through a specialized protozoan structure termed the cytostome, is poorly understood. Here, we present new data which, together with prior published work, strongly suggests that the anti-malarial action of mefloquine (MQ) is closely associated with the inhibition of hemoglobin endocytosis. In this proposal, we will test the hypothesis that MQ binds to a protein target that plays a key role in the parasite?s endocytosis of host erythrocyte hemoglobin. In Aim 1, we will synthesize a library of photoaffinity probes that are based on the MQ pharmacophore. Probe design will focus on the incorporation of ?all-in-one? moieties that enable the introduction of photoactivatable and affinity groups with minimal structural perturbation. Phenotypic and parasite viability assays will be employed to characterize probe potency and to verify retention of MQ?s mode of action. In Aim 2, these probes will be used to irreversibly label the protein target of MQ. Specificity of labeling will be assessed by competition with unmodified MQ and the characterization of purified enantiomers of selected probes. Putative targets will be affinity purified, identified by mass spectrometry, and validated using a suite of complementary approaches. These studies hold great promise to address the long-standing questions of the molecular mechanisms of hemoglobin endocytosis and of the mode of action of MQ, thereby identifying a potentially high-value anti-malarial target.